Animal husbandry constitutes the biggest portion of agricultural production in Taiwan, and brings handsome and stable income to Taiwanese farmers. However, fecal waste produced by livestock in large amount is not properly disposed and thus results in environmental pollution. According to the statistical information from the Council of Agriculture, Executive Yuan, Taiwan, the headcount of farmed hogs was 6,794,000 by the end of the year 2002. Assuming that each hog produces 6 Kg of fecal waste per day, there will be about 15,000,000 metric tons of hog fecal waste per year, plus the fecal waste from poultry. The serious environmental pollution resulting from such a large amount of fecal waste has become the major problem in Taiwanese animal husbandry. How to reduce the production of fecal waste without jeopardizing farmers' income is an important issue.
In addition to the aforementioned environmental issue, Taiwanese animal husbandry has to compete with imported livestock products since Taiwan joined the World Trade Organization (WTO) in 2002. How to reduce the production cost while enhancing the production rate has become another important issue. Generally speaking, the cost of feed constitutes 60 to 70% of the total production cost. Therefore, the key to reduce the production cost is to improve feed utilization. Effective ways include utilizing biotechnology to develop feed additives for enhancing the digestion of feed, and breeding livestock animals able to digest cellulose to reduce fecal production.
The quantity and quality of animal feces depend on the digestion and utilization of the components of feed by the animal. Generally speaking, livestock animals do not have the ability to synthesize digestive enzymes such as cellulases and phytases, and thus cannot effectively digest the cellulose and plant phosphorus in the feed. Such inability affects feed utilization and results in a waste of feed. In addition, the undigested nutrients are excreted from the animals and become a cause of environmental pollution. A common method for enhancing utilization of the nutrients in feed by animals is to supplement the feed with certain digestive enzymes produced on large scale by microorganisms. Recently, the cDNAs of several digestive enzymes have been screened out from fungal and bacterial cDNA libraries, and the enzymes have been produced on large scale by E. coli (Ye et al., 2001, Inter. J. Biochem. Biol. 33:87-94; and Zhang et al., 1998, Biotechnol. Lett. 20: 1001-1005; both hereby incorporated herein by reference). However, the production cost of this method is high. In addition, the supplementary digestive enzymes are usually destroyed in the animal's digestive tract by the native enzymes or gastric acid and thus cannot carry out their function.
Since the 1980s, the generation of transgenic animals not only allows us to study gene functions in vivo, but also provides a new way to improve the genetics of livestock animals. In the conventional breeding method, it takes tens of or even over a hundred years to improve the genetics of livestock animals. However, utilizing the techniques of genetic transformation, the same improvement can be achieved within one generation. In 1985, Hammer et al. first published their results in transgenic pigs, sheep and rabbits (Hammer et al., 1985, Nature 315:680-683; hereby incorporated herein by reference). Afterwards, researchers have utilized genes encoding growth factors from various animals (including cattle, mice and humans) to generate transgenic pigs carrying heterologous growth factor genes, hoping to increase lean meat in pigs and to shorten rearing time (Pursel and Rexroad, 1993, J. Anim. Sci. 71:10-19; hereby incorporated herein by reference). For example, Pursel et al. have constructed several heterologous growth factor genes under the control of the mouse metallothionein-I promoter (Pursel et al., 1989, Science 244:1281-1288; hereby incorporated herein by reference), and thereby the transcription of the heterologous genes can be induced and regulated effectively by the addition of trace amounts of zinc in the feed.
As known in the art, manipulation of animals (such as pigs) to alter and/or improve phenotypic characteristics (such as productivity or quality) requires the expression of heterologous genes in animal tissues. Such genetic manipulation therefore relies on the availability of means to drive and to control gene expression as required; for example, on the availability and use of suitable promoters which are effective in animals and which regulate gene expression so as to give the desired effect(s) in the transgenic animal. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, cell, tissue, animal or environment.
There is a continuing need in the art for high-level expression promoters, as well as promoters which are spatially defined in their expression patterns.